Gross chromosomal rearrangement (GCR) is a hallmark of cancer and DNA double- strand breaks (DSBs) constitute a major source of GCR. Many GCR events involve a replicative process, but the underlying mechanism is not clear. Based on the study in yeast, a DSB repair pathway called break-induced replication (BIR) is believed to be a major source of replicative GCR. Since the mechanistic study of BIR has been mostly carried out in yeast, a model system to study BIR mechanism and its role in suppressing genome instability in mammalian cells is highly in demand. We have established a novel EGFP-based BIR reporter, making it possible to systematically study BIR mechanisms and analyze its genetic components in mammalian cells. We hypothesize that BIR, on one hand, is involved in DSB repair to prevent chromosome breakages and help maintenance of genome stability, but on the other hand, is highly mutagenic and its dysregulation would contribute to genome instability. In this proposal, we will study how BIR is induced by replication and oncogenic stress and contributes to genome instability. We will also study the role and the underlying mechanism of replication helicases such as Pif1 and other replication proteins in BIR. We will decipher the role of ATR-mediated phosphorylation in promoting BIR and examine functional interplay of replication proteins with Rad51 at collapsed forks to initiate BIR for replication restart. Our study will shed light on how BIR is operated in mammalian cells and provide new mechanistic insight on the maintenance of genome integrity. It will also open new opportunities to explore the BIR pathway for cancer treatment.